GSM5603759: RNAseq-HL-E125-WT-GFP; Mus musculus; RNA-Seq
After sorting, cells were centrifuged for 5' at 400g at 4°C, supernatant was discarded and cells frozen at -80°C. Total RNA from 1,5 x10^5 cells was isolated using the RNeasy Micro Kit (QIAGEN, ID:74004) following manufacturer's instructions and then stored frozen at -80°C. Total RNA was quantified with a Qubit (fluorimeter from Life Technologies) and RNA integrity assessed with a Bioanalyzer (Agilent Technologies). The SMART-Seq v4 kit from Clontech was used for the reverse transcription and cDNA amplification according to manufacturer's specifications, starting with 5 ng of total RNA as input. 200 pg of cDNA were used for library preparation using the Nextera XT kit from Illumina. Library molarity and quality was assessed with the Qubit and Tapestation using a DNA High sensitivity chip (Agilent Technologies). Libraries were pooled at 2 nM and loaded for clustering on a Single-read Illumina Flow cell for an average of 35 mio reads / library. Reads of 50 bases were generated using the TruSeq SBS chemistry on an Illumina HiSeq 4000 sequencer.